anti p53 mutant  (Boster Bio)

Journal: Asian Pacific Journal of Cancer Prevention

Article Title: The Association between p53 Expression and Histopathology Grade of Astrocytoma

doi: 10.31557/apjcp.2025.26.7.2521

Figure Lengend Snippet: Figure 1. Immunohistochemistry Staining of p53 Mutant (High Power View, Magnification 400x). (A). Intense nuclear staining in immunopositive in ³10% of tumor cells was interpreted as positive (p53 mutant). (B). Weak nuclear staining was interpreted as negative as immunonegative (p53 wild-type)

Article Snippet: The research samples comprised Formalin-Fixed Paraffin-Embedded (FFPE) for Hematoxylin-Eosin (HE) and immunohistochemical staining with Anti-p53 mutant (M00001-3, Boster, Pleasanton, CA, USA).

Techniques: Immunohistochemistry, Staining, Mutagenesis

Journal: Cell reports. Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma.

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Figure 4. Detection of plasma-circulating mutant p53 in patients with DMG (A) Experimental scheme: biotinylated capture antibodies targeting total p53 protein (the WT and mutant form) are anchored to a PEG-streptavidin surface (a-p53). Plasma-circulating p53 protein molecules are captured on surface, followed by incubation with two distinct fluorescently labeled p53 antibodies: an antibody targeting all forms of p53 (‘‘total p53,’’ red) and an antibody specific to the mutant conformation of p53 (‘‘mutant p53,’’ green). (B) Representative TIRF images of the indicated p53 antibodies incubated on surfaces enriched for plasma-circulating p53 proteins. Scale bar applies to all images. (C) A cohort of 19 plasma samples of H3-K27M DMG patients was analyzed as described in (A) (13 samples harbor TP53 mutations and 6 harbor WT TP53). Principal component analysis (PCA) with the following input parameters: normalized counts of total p53 and mutant p53, and the ratio between mutant and total p53. Sample groups are color-coded according to known TP53 status; each dot represents one plasma sample. (D) The ratio between mutant and total p53 signal for each sample is shown. Each bar represents a sample, color-coded according to known TP53 status. Data are presented as the mean ± SD of 50 FOVs per sample. (E) Boxplot representation of the data in (D) grouped according to TP53 status (mutant p53 n = 13, WT-p53 n = 6). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.53 the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test. ***p value <0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#5499; RRID:AB_2797612 Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#4484; RRID:AB_10695884 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#12064; RRID:AB_2797813 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#13969; RRID:AB_2798355; Conjugated for this study Mono-Methyl-Histone H3 (Lys4) (D1A9) XP Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#5326; RRID:AB_10695148; Conjugated for this study Tri-Methyl-Histone H3 (Lys36) (D5A7) XP Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#4909; RRID:AB_1950412 Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#39030; RRID:AB_2799145 Histone H3 (K27M Mutant Specific) Rabbit mAb Cell Signaling Cat#74829; RRID:AB_2799861 Anti-p53 antibody [PAb 1801] Abcam Cat#ab28; RRID:AB_303312 Anti-p53 antibody [E47] - BSA and Azide free Abcam Cat#ab247264 Anti-p53 antibody [SP161] Abcam Cat#ab227655 Chemicals, peptides, and recombinant proteins Klenow Fragment (30/50 exo-) NEB Cat#M0212S T4 polynucleotide kinase NEB Cat#M0201L terminal deoxynucleotidyl transferase Enzymatics Cat#P7070L TetraSpeck beads Thermo Fisher Scientific Cat#T7279 Critical commercial assays Biotinylation Kit/Biotin Conjugation Kit (Fast, Type B) - Lightning-Link Abcam ab201796 Alexa FluorTM Antibody Labeling Kits Thermo Fisher Scientific Cat#A20181, Cat#A10237, Cat#A20186 CellTiter-Glo assay Promega Cat#G7572 QIAamp Circulating Nucleic Acid Kit QIAGEN Cat#55114 Experimental models: Cell lines HEK293 pInducer H3.3 K27M Furth et al.31 – HEK293 pInducer H3.3 wt Furth et al.31 – UMPED83 Koschmann lab, UM – Software and algorithms Cellprofiler Broad Institute RRID:SCR_007358 Original code This paper Zenodo: https://doi.org/10.5281/zenodo.14242990

Techniques: Clinical Proteomics, Mutagenesis, Incubation, Labeling

Journal: Cell reports. Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma.

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Figure 5. Single-molecule measurements of mutant nucleosomes as a potential proxy for tumor growth (A) Experimental scheme for parallel measure- ments of cell viability and H3-K27M-mutant nu- cleosomes released to culture media. Cells were plated as monolayer, and a day later media was replaced to initiate the experiment. Media was collected for single-molecule measurements and replaced to account for newly released nucleo- somes between examined time points. Viability measurements were conducted from a parallel culture. (B) Viability measurements and H3-K27M-H3K27ac single-molecule measurements are shown for un- treated culture (left) and 5 mM ONC201-treated culture (right). The drug was replenished every 2 days. For single-molecule measurements, mean ± SD of 40 FOVs for each time point is shown. For CellTiter-Glo measurements, mean ± SE of three technical repeats for each time point is shown. (C) Clinical features, initial MRI, and MRI of disease at clinical progression of DMG patient UMPED 128. (D–J) Analysis of serial plasma samples from pa- tients undergoing ONC201/206 treatment. Single- molecule measurements (normalized to first data point) of mutant nucleosomes (H3-K27M-H3K27ac) were performed on serial time point plasma sam- ples plotted along with tumor cross-sectional area according to MRI. Green bars: ONC201/206 mainline treatment. Purple bars: radiation treat- ment. Brown bars: other treatment modalities that are detailed in Table S1. Radiographic progression is marked with a black asterisk placed next to the corresponding MRI data point. Day 0 corresponds to the initiation of treatment. (K) Heatmap showing the correlation between the direction of change for the indicated features (see STAR Methods). For the majority of patients, tumor MRI measurements show the highest correlation with the single-molecule measurements of H3- K27M-H3K27ac in plasma. The correlation was calculated based on interpolated data points to account for measurements not taken at exactly the same time. See also mutant p53 and ctDNA data in Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#5499; RRID:AB_2797612 Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#4484; RRID:AB_10695884 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#12064; RRID:AB_2797813 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#13969; RRID:AB_2798355; Conjugated for this study Mono-Methyl-Histone H3 (Lys4) (D1A9) XP Rabbit mAb (Alexa Fluor 488 Conjugate) Cell Signaling Cat#5326; RRID:AB_10695148; Conjugated for this study Tri-Methyl-Histone H3 (Lys36) (D5A7) XP Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#4909; RRID:AB_1950412 Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Alexa Fluor 647 Conjugate) Cell Signaling Cat#39030; RRID:AB_2799145 Histone H3 (K27M Mutant Specific) Rabbit mAb Cell Signaling Cat#74829; RRID:AB_2799861 Anti-p53 antibody [PAb 1801] Abcam Cat#ab28; RRID:AB_303312 Anti-p53 antibody [E47] - BSA and Azide free Abcam Cat#ab247264 Anti-p53 antibody [SP161] Abcam Cat#ab227655 Chemicals, peptides, and recombinant proteins Klenow Fragment (30/50 exo-) NEB Cat#M0212S T4 polynucleotide kinase NEB Cat#M0201L terminal deoxynucleotidyl transferase Enzymatics Cat#P7070L TetraSpeck beads Thermo Fisher Scientific Cat#T7279 Critical commercial assays Biotinylation Kit/Biotin Conjugation Kit (Fast, Type B) - Lightning-Link Abcam ab201796 Alexa FluorTM Antibody Labeling Kits Thermo Fisher Scientific Cat#A20181, Cat#A10237, Cat#A20186 CellTiter-Glo assay Promega Cat#G7572 QIAamp Circulating Nucleic Acid Kit QIAGEN Cat#55114 Experimental models: Cell lines HEK293 pInducer H3.3 K27M Furth et al.31 – HEK293 pInducer H3.3 wt Furth et al.31 – UMPED83 Koschmann lab, UM – Software and algorithms Cellprofiler Broad Institute RRID:SCR_007358 Original code This paper Zenodo: https://doi.org/10.5281/zenodo.14242990

Techniques: Mutagenesis, Clinical Proteomics

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: Primers for cDNA sequencing of p53 gene.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: RT-PCR primers for p53 targets.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: Details of LFS patients from which fibroblast cells are taken.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques:

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: A Representative chromatograms showing TP53 heterozygosity at early passage (left panel) and at late passage (right panel). The adenine peak (arrows) is visible at late passage ( N = 4). B Western blot analysis of lysates from early (E), middle (M), and late (L) passage cells. Total p53 was detected with the DO-1 antibody, and mutp53 was detected with a mutp53–specific antibody. GAPDH was used as a loading control (bottom lane) ( N = 4). C Immunofluorescence analysis of early and late passage LFS fibroblasts. Mutp53–specific antibody (green) was used for LOH confirmation and nuclei were stained with DAPI (blue). (Scale bar, 20 μm.) Quantification of mutp53 staining was done using ImageJ ( N = 4, n > 200). D Brightfield microscopic images of LFS fibroblasts undergoing senescence, as detected by β-gal assay; blue indicates senescent cells. Wild-type (WT) p53 fibroblasts at early (P12) and late (P21) and LFS fibroblasts at early and late passage were analyzed ( N = 4). E Immunofluorescence analysis was performed on fibroblasts incubated with FITC-labeled mutp53–specific antibody (green), and yH2AX was detected by red fluorescence. Nuclei were stained with DAPI (blue). (Scale bar, 10 μm.) Quantification of DNA damage marker yH2AX was done using ImageJ ( N = 4, n > 200). * p < 0.05, ** p < 0.01, *** p < 0.001. Two-tailed unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Incubation, Labeling, Fluorescence, Marker, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: A Western blot analysis of lysates from WT, early passage LFS, late passage LFS, and late passage LFS cells treated with pCAP-250 or with a scrambled peptide. Total p53 was detected with the DO-1 antibody, and mutp53 was detected with a mutp53–specific antibody. GAPDH served as loading control. Data was quantified using ImageJ ( N = 4). B Cells were plated in 10-cm dishes at a density of 100,000 cells/dish and then treated with either pCAP-250 or a scrambled peptide or left untreated as a control (NTC). The cells were counted by brightfield imaging at 4× after 72 h ( N = 4). C Immunofluorescence analysis was performed on nontreated late passage cells and late passage cells treated with pCAP-250. Ki67-specific antibody was labeled with FITC, and the nuclei were stained with DAPI (blue). (Scale bar, 20 μm) Images were taken at 20X and staining was quantified using ImageJ ( N = 4, n > 150). D Brightfield microscopic images of cells undergoing senescence, as detected by β-gal assay. Blue represents senescent cells. WT and LFS fibroblasts were imaged at early and late passages, along with late passage LFS cells treated with pCAP-250, and senescent cells were quantified ( N = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. Two-tailed unpaired Student’s -test.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques: Western Blot, Control, Imaging, Immunofluorescence, Labeling, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

doi: 10.1038/s41418-024-01307-4

Figure Lengend Snippet: A Comet assay was performed on early passage, late passage, and late passage plus pCAP-250 cultures. Data was acquired using epifluorescence imaging (Scale bar, 50 μm.) and Tail length was quantified using ImageJ ( N = 4, n > 200). B RT-qPCR was performed on early passage cells, late passage cells and late passage cells treated with pCAP-250, to quantify the expression of genes encoding the DNA repair enzymes XPC, MSH2 and FEN1, which are regulated by WT p53. Cisplatin served as a positive control to induce the expression of these genes by WTp53 activation ( N = 4). C CPD lesions were detected in late passage cells 53528 treated with increasing doses of UV radiation. The cells were incubated in culture medium for 24 h, and lesions were counted and quantified by ImageJ ( n = 50). D Spectral karyotyping was performed on LFS early passage, late passage, and late passage cells treated with pCAP-250 ( n = 10). Percentages of cells with chromosomal aberrations were calculated. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. Two-tailed unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used: DO-1 p53, β-actin (Santa Cruz Biotechnology, Dallas, TX USA; sc-47778), mutant p53 (Y5; Abcam, Cambridge, UK).

Techniques: Single Cell Gel Electrophoresis, Imaging, Quantitative RT-PCR, Expressing, Positive Control, Activation Assay, Incubation, Two Tailed Test